Ammonia is associated with liver-related complications and predicts mortality in acute-on-chronic liver failure patients

The relationship between ammonia and liver-related complications (LRCs) in acute-on-chronic liver failure (ACLF) patients is not clearly established. This study aimed to evaluate the association between ammonia levels and LRCs in patients with ACLF. The study also evaluated the ability of ammonia in predicting mortality and progression of LRCs. The study prospectively recruited ACLF patients based on the APASL definition from the ACLF Research Consortium (AARC) from 2009 to 2019. LRCs were a composite endpoint of bacterial infection, overt hepatic encephalopathy (HE), and ascites. A total of 3871 cases were screened. Of these, 701 ACLF patients were enrolled. Patients with LRCs had significantly higher ammonia levels than those without. Ammonia was significantly higher in patients with overt HE and ascites, but not in those with bacterial infection. Multivariate analysis found that ammonia was associated with LRCs. Additionally, baseline arterial ammonia was an independent predictor of 30-day mortality, but it was not associated with the development of new LRCs within 30 days. In summary, baseline arterial ammonia levels are associated with 30-day mortality and LRCs, mainly overt HE and ascites in ACLF patients.


Statistical analysis
Categorical variables were presented as numbers and percentages and compared between groups using the Pearson Chi-square or Fisher's exact test.Continuous variables were analyzed for normality using the Shapiro-Wilk test.Normally distributed data were expressed as mean (standard deviation) with group means comparisons using the independent t-test, while non-normally distributed data were expressed as median (interquartile range) with group comparison testing using the Mann-Whitney U test.Multivariate logistic regression was done to assess the association with LRCs at baseline.Cox regression analyses identified potential factors associated with 30-day mortality.The Kaplan-Meier graph and survival predictions were compared using the Log-rank test.Area under the receiver operating characteristics (AUROC), and Youden's index were analyzed to identify the optimal ammonia cutoffs for 30-day mortality.Comparison of AUROC was done by the Medcalc Software (Belgium) using Delong's technique.Statistical analysis was performed using the Statistical Package for the Social Sciences (SPSS) software, Version 23 (IBM Corporation, Armonk, NY, USA).Two-sided p-values less than 0.05 were considered statistically significant.

Patient baseline characteristics
A total of 3871 patients with ACLF were screened.Of these, 701 patients with ACLF were enrolled.The most common reason for exclusion was a lack of ammonia presence.Baseline characteristics of patients who were included and excluded were shown in the Supplementary Table 1.Patients who were excluded had higher rates of LRCs and alanine aminotransferase (ALT), but a lower proportion of ACLF grade II-III, total bilirubin (TB), INR, creatinine, and lactate levels than those who were included.In patients who were enrolled, most of the patients were male (85.6%, n = 600), with a median age of 43 (IQR: 37-52) years.The most common cause of chronic liver disease or cirrhosis was alcohol-related liver disease (84.2%, n = 590), followed by hepatitis B virus infection (15.8%, n = 111).The median Model for End-Stage Liver Disease (MELD), Sequential Organ Failure Assessment (SOFA), and AARC scores were 29.0 (IQR: 24.3-35.0),8 (IQR: 6-10) and 9 (IQR: 8-11), respectively.At baseline, 127 patients (18.1%) had ACLF grade I, 364 (51.9%) had ACLF grade II, and 210 (30.0%) had ACLF grade III.In addition, 427 patients (60.9%) had at least one LRC, 346 (49.4%) had ascites, 276 (39.4%) had overt HE, and 245 (35.0%) had bacterial infections at admission.Baseline patient characteristics according to each LRC, overt HE, ascites, and bacterial infection were analyzed and shown in the Supplementary Table 2. ACLF with overt HE, ascites, or bacterial infection were sicker, as evidenced by higher MELD score than those without these complications.Results from the univariate analysis revealed that total WBC, TB, INR, and ammonia levels were associated with LRCs.Multivariate analysis showed that ammonia levels (OR = 1.011, 95%CI 1.009-1.014,p < 0.001) was independently associated with LRCs (Table 2).

Factor associated with 30-day mortality
Overall, the 30-day mortality rate was 49.9% (n = 350).ACLF patients who died within 30 days after hospitalization were older and had a proportional higher incidence of alcohol-related liver disease, baseline LRCs, and ACLF grade III.Higher baseline MELD, SOFA, and AARC scores were also reported compared to patients who survived the 30-day period.Baseline ammonia (p < 0.001) and lactate (p < 0.001) was significantly lower in survivors compared to non-survivors (Supplementary Table 3).

Discussion
The current study investigated the association between arterial ammonia levels and LRCs in patients with ACLF.We also assessed the potential role of arterial ammonia in predicting mortality and development of LRCs during the 30 days after hospitalization.Our main finding showed that baseline ammonia levels are associated with LRC, particularly overt HE, and ascites.In addition, arterial ammonia is a potential predictor of 30-day mortality and LRC development.The study findings have highlighted the additional role of ammonia in detecting and predicting poor clinical outcomes other than HE in ACLF patients.Hyperammonemia is traditionally described as the central contributor of HE development.It is also associated with non-neurological organ injury.Ammonia exerts multiorgan dysfunction by promoting hepatocyte death, activation of hepatic stellate cells, and producing neutrophil phagocytic impairment, resulting in liver inflammation, fibrosis, portal hypertension, and increased risk for infection [17][18][19] .Tranah et al. demonstrated that ammonia independently predicts 1-year mortality and LRCs such as bacterial infection, variceal bleeding, ascites,  11 .In addition, ammonia outperforms Child-Pugh and MELD scores in predicting complications.Balcar et al. recently showed that ammonia is linked to liver-related death, liver dysfunction, and portal hypertension severity in clinically stable outpatients with advanced chronic liver disease 10 .In sicker patients, such as those with ACLF, ammonia is associated with mortality 8,12,20,21 , severity of ACLF 12 , and an increased risk of organ failure 12,21 .Consistent with the current study's findings, ammonia is independently associated with the severity of ACLF and 30-day mortality.To our knowledge, the study presents new evidence that ammonia is also independently related to the presence of LRCs, not only overt HE but also ascites in patients with ACLF.Although the study did not demonstrate the association between bacterial infection and high ammonia levels.This finding should be considered in the context that seventy percent (n = 172/245) of patients with bacterial infection had more than one liver-related complication.This could potentially impact the relationship between this complication and ammonia level.Further analysis in ACLF without baseline LRCs found that ammonia level predicts the new onset of LRC development within 30 days after admission.These findings support the additional prognostic role of ammonia in detecting and predicting complications in ACLF patients.However, the current study found that baseline ammonia levels ≥ 150 µmol/L was associated with 30-day mortality, but the AUROC was fair and had low sensitivity.
There is an unmet need for a universal definition of ACLF.Three main definitions from different consortiums around the world have been proposed from the APASL AARC, European Association for the Study of the Liver-Chronic Liver Failure (EASL-CLIF) consortium 22 , and the North American Consortium for the Study of End-Stage Liver Disease (NACSELD) 23 .Organ failure is one of the key differences among the three guidelines.APASL AARC requires hepatic failures, EASL-CLIF requires a combination of hepatic and extrahepatic organ failure, and NACSELD needs at least two extrahepatic organ failures.ACLF is characterized by acute hepatic insult presenting as jaundice and coagulopathy, followed by ascites and/or encephalopathy within 4 weeks in patients with prior chronic liver disease or cirrhosis by APASL guidelines 14 .The feature of acute decompensation indicates significant hepatic deterioration, supporting the importance of hepatic failure in these criteria.ACLF precipitated by hepatic insults is distinct from that caused by extrahepatic insults 24 .The presence of LRC in ACLF may indicate prominent portal hypertension.Development of LRCs is associated with high 6-week mortality and poor outcomes in patients with ACLF 25 .In addition, hepatic decompensation indicates poor reversibility of ACLF syndrome 26 .According to this study's findings, ammonia may guide physicians in managing these complications.
There are some limitations in this study.First, there could be discrepancies in technical concerns with ammonia testing among centers, even though investigators checked the test's reliability before entering data.Strict sample handling and processing requirements were the possible explanations for the differences in ammonia levels among testing sites 27 .Ammonia level is affected by various factors such as the centrifugation process, blood drawing tourniquet technique, transportation, and hemolysis.Second, the study only enrolled participants with alcohol-related liver disease and hepatitis B virus, since these are two common causes of chronic liver disease in the Asia-Pacific region.Third, variceal bleeding was not included in the list of liver-related complications due to the inaccessibility of this data in the majority of the hospitals.Fourth, there were a small number of patients without baseline LRCs who developed LRCs during the 30 days of follow-up, which might affect the result's validity.In addition, the management of ACLF relied on the decision-making of physicians in each hospital, which could impact the outcome of patients.If the patients were discharged earlier than day 30, they were re-evaluated clinical information at the outpatient clinic on the 30th day after enrollment.Ascites was assessed using abdominal imaging or clinical findings according to the attending physician's decision at each hospital.Therefore, patients with grade 1 ascites might be overlooked if they were not evaluated by ultrasound.
In conclusion, baseline arterial ammonia levels demonstrated an association with 30-day mortality and LRCs, encompassing not only overt HE but also ascites in patients with ACLF.

Table 2 .
Factors related to liver-related complications at admission.ALT; alanine aminotransferase, TB; total bilirubin, WBC; white blood cell count.WBC, TB, INR, and ammonia were included in the multivariate analysis.